Deposition Of Matrix Proteins Research Articles

Loss of PTPN21 disrupted mitochondrial metabolic homeostasis and aggravated experimental pulmonary fibrosis

Idiopathic pulmonary fibrosis (IPF) is a high-mortality lung disease with unclear pathogenesis. Convincing evidence suggests that an imbalance in mitochondrial homeostasis resulting from repeated injury to alveolar epithelial type 2 cells (AEC2) underlies IPF. Non-receptor protein tyrosine phosphatase 21 (PTPN21) performs various functions in cancer; however, its role in IPF has not been studied. This study aimed to investigate the role of PTPN21 in lung fibrosis. The experimental results showed that loss of PTPN21 exacerbated lung fibrosis by increasing cell numbers in bronchoalveolar lavage fluid, lung hydroxyproline content, and extracellular matrix protein expression of fibronectin and α-smooth muscle actin (α-SMA) in bleomycin-challenged mouse lungs. In A549 cells (AEC2), knockdown of PTPN21 suppressed focal adhesion and migration, reduced mitochondrial fission and increased fusion, increased the level of mitochondrial superoxide, decreased mitochondrial membrane potential and ATP levels. Simultaneously, knockdown of PTPN21 impaired autophagy, and increased intracellular reactive oxygen species levels. Treatment of fibroblasts (MRC-5) and primary human lung fibroblasts (PHLF)) with the supernatant from PTPN21-knockdown A549 cells increased the expression of fibronectin, collagen 1 and α-SMA. Conversely, overexpression of PTPN21 in A549 cells produced opposite effects. However, treatment of MRC-5 and PHLF with the supernatant from PTPN21-overexpressing A549 cells only slightly reduced the expression of fibronectin, collagen 1 in MRC-5 cells, but did not change the expression of α-SMA. In summary, this study revealed that the loss of PTPN21 in epithelial cells disrupted mitochondrial metabolic homeostasis, leading to epithelial cell inactivation and increased the deposition of extracellular matrix proteins in fibroblasts, thereby exacerbating experimental pulmonary fibrosis.

Diagnostic potential of genomic blood biomarkers of pulmonary fibrosis in a prospective cohort.

Fibrotic interstitial lung diseases (ILDs) result from excessive deposition of extracellular matrix (ECM) proteins in the lung, causing irreversible damage to the lung architecture. Clinical management of ILDs differs depending on the diagnosis, but differentiation between subtypes can be difficult and better clinical biomarkers are needed. In this study, we use a 166-gene NanoString assay to investigate whether there are ILD subtype-specific transcripts in whole blood. We identified one transcript, killer cell lectin like receptor 1 (KLRF1), as differentially expressed between idiopathic pulmonary fibrosis (IPF) and systemic sclerosis-associated ILD (SSc-ILD), and identified two transcripts (VCAN, LTK) associated with IPF expression against other ILD subtypes. These findings were validated by examining their expression in ILD lung, with KLRF1 expression significantly higher in SSc-ILD compared to IPF and hypersensitivity pneumonitis (HP) samples. Taken together, this pilot study provides support for the use of the peripheral transcriptome in identifying diagnostic biomarkers of ILD with biological relevance.

The role of fibromodulin in myocardial fibrosis in a diabetic cardiomyopathy rat model.

Diabetic cardiomyopathy (DCM) is pathologically characterized by excessive deposition of extracellular matrix proteins, leading to myocardial fibrosis. Fibromodulin (Fmod) plays a crucial role in the pathogenesis of fibrotic diseases. However, the role and mechanism of Fmod in DCM-related myocardial fibrosis remain unclear. In the present study, we established a DCM rat model and an in vitro model of rat primary cardiac fibroblasts (RPCFs) exposed to high glucose. We assessed mRNA and protein expression levels of Col1a1, Col3a1, α-SMA and Fmod in both models. Fmod-overexpressing (ov-Fmod) and Fmod-knockdown (si-Fmod) rat cardiac fibroblasts (RCFs) were generated. Subsequently, whole RNA sequencing was conducted on ov-Fmod RCFs. The gene Col15a1 was evaluated in the DCM rat and all cell models. The correlation between plasma levels of Fmod and Col15a1 in DCM rat models was assessed. Transcription and protein levels of Fmod, Col1a1, Col3a1 and α-SMA were significantly elevated in DCM rat hearts and RPCFs. In ov-Fmod RCFs, fibrosis markers were similarly increased, except for Col3a1, which decreased. The Col1a1/Col3a1 ratio was elevated. Conversely, knocking down Fmod yielded opposite results. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analyses indicated that Fmod participates in multiple fibrosis-related pathways, affecting Col15a1. Expression of Col15a1 was significantly decreased in all models, compared to controls, except in si-Fmod RCFs. Importantly, Col15a1 and Fmod in plasma exhibited an inverse relationship in DCM. In summary, Fmod is implicated in DCM, with Fmod overexpression downregulating Col15a1 and increasing the Col1a1/Col3a1 ratio. This mechanism may influence diastolic heart failure in DCM by modulating myocardial stiffness and elasticity.

Influence of metalloproteinase-3 (-1171 5A>6A) polymorphism on periportal fibrosis in patients with schistosomiasis mansoni, Pernambuco, Brazil

In schistosomiasis mansoni (SM), periportal fibrosis (PPF) arises due to an inflammatory response exacerbated by parasite eggs in the intrahepatic portal space, culminating in the deposition of collagen and extracellular matrix proteins. This fibrosis results from a remodeling process of the extracellular matrix, in which metalloproteinases play a significant role. The study evaluated the association between MMP-3 polymorphism (-1171 5A>6A) (rs 3025058) and sociodemographic factors with PPF in individuals with SM. This is an analytical cross-sectional study involving 242 individuals infected with S. mansoni, of these 122 were diagnosed with hepatosplenic form (HS) and 119 hepatointestinal form (HI), all from the state of Pernambuco, Brazil. Polymerase chain reaction with restriction enzyme digestion (Psyl) was used to determine the MMP-3 polymorphism (-1171 5A>6A). There was a significant association between the male gender and the HS form (OR = 1.7623 95% CI [1.0481–2.9631]; p-value = 0.0439) as well as individuals aged over 41, also had a greater chance of developing this clinical form of the disease (OR = 2.8299; 95% CI [1.5211–5.2650]; p-value = 0.0014), with greater emphasis on individuals over 61 years old (OR= 8.5541; 95% CI [3.6895–19.8326], p-value= 0.0000). There was no statistically significant association between the MMP-3 polymorphism (-1171 5A>6A) between the clinical groups (5A6A CI [0.7144–1.9879] p-value 0.5882 5A5A CI [0.0912–2.9231] p-value 0.7331 5A6A / 5A5A CI [0.6904–1.8937] p-value 0.6949). In conclusion, the results showed no association between the MMP-3 polymorphism (-1171 5A>6A) and the development of PPF. In addition, males, and age over 41 were predictive factors for the HS form of the disease in this Brazilian population.

Molecular Interplay in Cardiac Fibrosis: Exploring the Functions of RUNX2, BMP2, and Notch.

Cardiac fibrosis, characterized by the excessive deposition of extracellular matrix proteins, significantly contributes to the morbidity and mortality associated with cardiovascular diseases. This article explores the complex interplay between Runt-related transcription factor 2 (RUNX2), bone morphogenetic protein 2 (BMP2), and Notch signaling pathways in the pathogenesis of cardiac fibrosis. Each of these pathways plays a crucial role in the regulation of cellular functions and interactions that underpin fibrotic processes in the heart. Through a detailed review of current research, we highlight how the crosstalk among RUNX2, BMP2, and Notch not only facilitates our understanding of the fibrotic mechanisms but also points to potential biomolecular targets for intervention. This article delves into the regulatory networks, identifies key molecular mediators, and discusses the implications of these signaling pathways in cardiac structural remodeling. By synthesizing findings from recent studies, we provide insights into the cellular and molecular mechanisms that could guide future research directions, aiming to uncover new therapeutic strategies to manage and treat cardiac fibrosis effectively.

Selective activation of PPARα by pemafibrate mitigates peritoneal inflammation and fibrosis through suppression of NLRP3 inflammasome and modulation of inflammation

Peritoneal inflammation and fibrosis remain major challenges to the long-term maintenance of peritoneal dialysis. Pemafibrate, a selective peroxisome proliferator-activated receptor α (PPARα) modulator, has been implicated in the management of fibrosis-related disorders. We investigated whether pemafibrate ameliorates peritoneal inflammation and fibrosis and explored the underlying mechanisms in mice with methylglyoxal (MGO)-induced peritoneal fibrosis (MGO mice). MGO mice exhibited peritoneal fibrosis with increased expression of mesenchymal markers, transforming growth factor-β1 (TGF-β1), and substantial deposition of extracellular matrix (ECM) proteins. Additionally, MGO mice exhibited peritoneal inflammation as indicated by elevated tumor necrosis factor-α expression and macrophage infiltration in peritoneal tissue. These effects were mitigated by pemafibrate treatment, which also restored peritoneal membrane function. Furthermore, pemafibrate promoted anti-inflammatory macrophage polarization in both mice and THP-1 cells. In human peritoneal mesothelial cells (HPMCs), pemafibrate effectively inhibited interferon-γ-induced production of TGF-β1 and ECM while suppressing the proinflammatory cytokines nuclear factor-κB (NF-κB) and activator protein 1. The NF-κB inhibitory effect of pemafibrate involved stabilization of the NF-κB inhibitory protein IkBα. Notably, pemafibrate hindered activation of the NLR family pyrin domain containing 3/caspase-1 axis in interferon-γ-stimulated THP-1 cells. These findings suggest that pemafibrate ameliorates peritoneal inflammation and fibrosis, making it a promising candidate for peritoneal fibrosis therapy.

Glycosaminoglycan Concentration and Sulfation Patterns of Biohybrid Polymer Matrices Direct Microvascular Network Formation and Stability

AbstractComplexation with sulfated glycosaminoglycans (GAGs) regulates the signaling of pro‐angiogenic growth factors in vivo. To use this principle in modulating microvascular network formation and stability in vitro, a library of biohybrid hydrogels containing heparin, a highly sulfated GAG, or its selectively desulfated derivatives, is prepared. The hydrogels are applied to systematically investigate the effects of GAG concentration and sulfation patterns on embedded cultures of human umbilical vein endothelial cells (HUVECs) and cocultures of HUVECs with mesenchymal stromal cells (MSCs). Formation and morphology of microvascular networks, stromal cell expansion, and extracellular matrix (ECM) protein deposition are found to be significantly influenced by the hydrogel's GAG concentrations and sulfation patterns, presumably through differences in the resulting availability and bioactivity of GAG‐binding growth factors. In particular, cocultures in hydrogels with either low heparin concentration (500 µm) or higher concentrations of heparin desulfated at the 6O or N position resulted in the formation of dense, stable microvascular networks. The respective conditions also displayed enhanced stromal cell proliferation and ECM protein deposition, leading to increased matrix stiffness. Therefore, precisely tuning the affinity of biohybrid materials for pro‐angiogenic factors effectively enhances the vascularization of engineered tissue constructs in vitro.

Testicular fibrosis pathology, diagnosis, pathogenesis, and treatment: A perspective on related diseases.

Testicular fibrosis is a chronic and progressive condition characterized by the excessive deposition of extracellular matrix proteins. This process leads to fibrotic remodeling, damage to testicular tissue, and the irreversible loss of male reproductive function. However, there is currently a lack of comprehensive reviews systematically elucidating the pathology, diagnosis, pathogenesis, and treatment of testicular fibrosis from the perspectives of different related diseases. This review addresses these aspects of testicular fibrosis, with a particular emphasis on elucidating the underlying mechanisms of testicular cells. It provides insights that can be relevant for future research and clinical interventions.

Ablation of LRP6 in alpha-smooth muscle actin-expressing cells abrogates lung inflammation and fibrosis upon bleomycin-induced lung injury.

Low-density lipoprotein receptor-related protein 6 (LRP6) is a receptor for Wnt ligands. Tissue fibrosis is a progressive pathological process with excessive extracellular matrix proteins (ECM) deposition. Myofibroblasts, identified by alpha-smooth muscle actin (αSMA) expression, play an important role in tissue fibrosis by producing ECM production. Here we found that Wnt antagonist Dickkopf1 (DKK1) induced gene expressions associated with inflammation and fibrosis in lung fibroblasts. We demonstrated that genetic deletion of LRP6 in αSMA-expressing cells using Acta2 -cre Lrp6 fl/fl ( Lrp6 AKO ) mice abrogated bleomycin (BLM)-induced lung inflammation and fibrosis phenotype, suggesting an important role of LRP6 in modulating inflammation and fibrotic processes in the lung. Our results highlight the crucial role of LRP6 in fibroblasts in regulating inflammation and fibrosis upon BLM-induced lung injury.

MR Molecular Image Guided Treatment of Pancreatic Cancer with Targeted ECO/miR-200c Nanoparticles in Immunocompetent Mouse Tumor Models.

Pancreatic ductal adenocarcinoma (PDAC) is characterized by desmoplasia due to increased deposition of extracellular matrix (ECM) proteins. This work investigates the efficacy of targeted ECO/miR-200c nanoparticles (ELNP) on ECM remodeling in PDAC and tumor proliferation with MR molecular imaging (MRMI) with MT218 in immunocompetent mouse models. The miR-200c mediated regulation of EMT markers was measured in PDAC cells in vitro. Wild-type mice bearing mutated KRAS-driven KPC subcutaneous or orthotopic tumors were dosed weekly with RGD-ELNP/miR-200c at 1mg-RNA/kg for a total of 4 doses. We utilized MT218-MRMI to non-invasively monitor the alteration of tumor ECM EDN-FN levels by miR-200c and tumor response to the treatment. The changes were also validated by posthumous histopathology. Transfection of PDAC cells with ELNP/miR-200c downregulated the expression of FN1 and EDB-FN and some mesenchymal markers, inhibiting 3D spheroid formation and migration of KPC PDAC cells. RGD-ELNP/miR-200c treatment resulted in significant signal reduction in the MT218 enhanced MRMI images of both subcutaneous and orthotopic KPC tumors compared to those prior to treatment and treated with a non-specific control. MT218-MRMI results were suggestive of EDB-FN downregulation in tumors, which was later confirmed by immunohistochemistry. Tumor growth in subcutaneous tumors was significantly attenuated with RGD-ELNP/miR-200c and was an observed trend in orthotopic tumors. Substantial necrosis and remodeling were observed in both models treated with RGD-ELNP/miR-200c based on H&E staining. These results demonstrate the feasibility of RGD-ELNP/miR-200c in modulating PDAC ECM and restraining tumor growth and the utility of MT218-MRMI for non-invasively monitoring miR-200c efficacy.

Bone morphogenetic protein-3 is a negative regulator of transforming growth factor beta and fibrosis

Fibrosis results in one-third of all deaths globally and is a major healthcare challenge. Fibrosis is scarring caused by the excess deposition of extracellular matrix proteins by fibroblasts. Inhibition of pathways downstream of transforming growth factor β (TGF-β) a pluripotent growth factor, has potent antifibrotic effects in different organs. Here we show that loss of bone morphogenetic protein (BMP-3) is a feature of kidney fibrosis, independent of the initiating injury, suggesting loss of this cytokine is a core fibrotic mechanism. TGF-β decreased BMP3 expression in human fibroblasts is possibly a feed-forward loop that contributes to increased and sustained TGF-β activity. Recombinant human BMP-3 reduced TGF-β induced fibroblast contraction, migration and invasion, pathways that lead to scarring and tissue stiffening. BMP-3 reduced TGF-β stimulated collagen cross-linking, and Ox-LDL receptor 1, a regulator of collagen deposition. BMP-3 inhibited TGF-β stimulated lysyl oxidase activity. Lysyl oxidase mediated collagen cross-linking is a critical process in TGF-β induced fibrosis. We propose that BMP-3 alters fibroblast responses to TGF-β, shifting the balance from fibrosis to repair. Recombinant human BMP-3 shows promise for development as a novel therapeutic for fibrosis.

Liver Fibrosis: From Basic Science towards Clinical Progress, Focusing on the Central Role of Hepatic Stellate Cells.

The burden of chronic liver disease is globally increasing at an alarming rate. Chronic liver injury leads to liver inflammation and fibrosis (LF) as critical determinants of long-term outcomes such as cirrhosis, liver cancer, and mortality. LF is a wound-healing process characterized by excessive deposition of extracellular matrix (ECM) proteins due to the activation of hepatic stellate cells (HSCs). In the healthy liver, quiescent HSCs metabolize and store retinoids. Upon fibrogenic activation, quiescent HSCs transdifferentiate into myofibroblasts; lose their vitamin A; upregulate α-smooth muscle actin; and produce proinflammatory soluble mediators, collagens, and inhibitors of ECM degradation. Activated HSCs are the main effector cells during hepatic fibrogenesis. In addition, the accumulation and activation of profibrogenic macrophages in response to hepatocyte death play a critical role in the initiation of HSC activation and survival. The main source of myofibroblasts is resident HSCs. Activated HSCs migrate to the site of active fibrogenesis to initiate the formation of a fibrous scar. Single-cell technologies revealed that quiescent HSCs are highly homogenous, while activated HSCs/myofibroblasts are much more heterogeneous. The complex process of inflammation results from the response of various hepatic cells to hepatocellular death and inflammatory signals related to intrahepatic injury pathways or extrahepatic mediators. Inflammatory processes modulate fibrogenesis by activating HSCs and, in turn, drive immune mechanisms via cytokines and chemokines. Increasing evidence also suggests that cellular stress responses contribute to fibrogenesis. Recent data demonstrated that LF can revert even at advanced stages of cirrhosis if the underlying cause is eliminated, which inhibits the inflammatory and profibrogenic cells. However, despite numerous clinical studies on plausible drug candidates, an approved antifibrotic therapy still remains elusive. This state-of-the-art review presents cellular and molecular mechanisms involved in hepatic fibrogenesis and its resolution, as well as comprehensively discusses the drivers linking liver injury to chronic liver inflammation and LF.

GPR176 promotes fibroblast-to-myofibroblast transition in organ fibrosis progression

Fibrosis is characterized by excessive deposition of extracellular matrix proteins, particularly collagen, caused by myofibroblasts in response to chronic inflammation. Although G protein-coupled receptors (GPCRs) are among the targets of current antifibrotic drugs, no drug has yet been approved to stop fibrosis progression. Herein, we aimed to identify GPCRs with profibrotic effects. In gene expression analysis of mouse lungs with induced fibrosis, eight GPCRs were identified, showing a >2-fold increase in mRNA expression after fibrosis induction. Among them, we focused on Gpr176 owing to its significant correlation with a myofibroblast marker α-smooth muscle actin (αSMA), the profibrotic factor transforming growth factor β1 (TGFβ1), and collagen in a human lung gene expression database. Similar to the lung fibrosis model, increased Gpr176 expression was also observed in other organs affected by fibrosis, including the kidney, liver, and heart, suggesting its role in fibrosis across various organs. Furthermore, fibroblasts abundantly expressed Gpr176 compared to alveolar epithelial cells, endothelial cells, and macrophages in the fibrotic lung. GPR176 expression was unaffected by TGFβ1 stimulation in rat renal fibroblast NRK-49 cells, whereas knockdown of Gpr176 by siRNA reduced TGFβ1-induced expression of αSMA, fibronectin, and collagen as well as Smad2 phosphorylation. This suggested that Gpr176 regulates fibroblast activation. Consequently, Gpr176 acts in a profibrotic manner, and inhibiting its activity could potentially prevent myofibroblast differentiation and improve fibrosis. Developing a GPR176 inverse agonist or allosteric modulator is a promising therapeutic approach for fibrosis.

Inhibition of tartrate-resistant acid phosphatase 5 can prevent cardiac fibrosis after myocardial infarction

BackgroundMyocardial infarction (MI) leads to enhanced activity of cardiac fibroblasts (CFs) and abnormal deposition of extracellular matrix proteins, resulting in cardiac fibrosis. Tartrate-resistant acid phosphatase 5 (ACP5) has been shown to promote cell proliferation and phenotypic transition. However, it remains unclear whether ACP5 is involved in the development of cardiac fibrosis after MI. The present study aimed to investigate the role of ACP5 in post-MI fibrosis and its potential underlying mechanisms.MethodsClinical blood samples were collected to detect ACP5 concentration. Myocardial fibrosis was induced by ligation of the left anterior descending coronary artery. The ACP5 inhibitor, AubipyOMe, was administered by intraperitoneal injection. Cardiac function and morphological changes were observed on Day 28 after injury. Cardiac CFs from neonatal mice were extracted to elucidate the underlying mechanism in vitro. The expression of ACP5 was silenced by small interfering RNA (siRNA) and overexpressed by adeno-associated viruses to evaluate its effect on CF activation.ResultsThe expression of ACP5 was increased in patients with MI, mice with MI, and mice with Ang II-induced fibrosis in vitro. AubipyOMe inhibited cardiac fibrosis and improved cardiac function in mice after MI. ACP5 inhibition reduced cell proliferation, migration, and phenotypic changes in CFs in vitro, while adenovirus-mediated ACP5 overexpression had the opposite effect. Mechanistically, the classical profibrotic pathway of glycogen synthase kinase-3β (GSK3β)/β-catenin was changed with ACP5 modulation, which indicated that ACP5 had a positive regulatory effect. Furthermore, the inhibitory effect of ACP5 deficiency on the GSK3β/β-catenin pathway was counteracted by an ERK activator, which indicated that ACP5 regulated GSK3β activity through ERK-mediated phosphorylation, thereby affecting β-catenin degradation.ConclusionACP5 may influence the proliferation, migration, and phenotypic transition of CFs, leading to the development of myocardial fibrosis after MI through modulating the ERK/GSK3β/β-catenin signaling pathway.

The hepatocyte epidermal growth factor receptor (EGFR) pathway regulates the cellular interactome within the liver fibrotic niche.

Liver fibrosis is the consequence of chronic liver injury in the presence of an inflammatory component. Although the main executors of this activation are known, the mechanisms that lead to the inflammatory process that mediates the production of pro-fibrotic factors are not well characterized. Epidermal growth factor receptor (EGFR) signaling in hepatocytes is essential for the regenerative processes of the liver; however, its potential role in regulating the fibrotic niche is not yet clear. Our group generated a mouse model that expresses an inactive truncated form of the EGFR specifically in hepatocytes (ΔEGFR mice). Here, we have analyzed the response of WT and ΔEGFR mice to chronic treatment with carbon tetrachloride (CCl4), which induces a pro-inflammatory and fibrotic process in the liver. The results indicated that the hallmarks of liver fibrosis were attenuated in CCl4-treated ΔEGFR mice when compared with CCl4-treated WT mice, coinciding with a faster resolution of the fibrotic process and ameliorated damage. The absence of EGFR activity in hepatocytes induced changes in the pattern of immune cells in the liver, with a notable increase in the population of M2 macrophages, more related to fibrosis resolution, as well as in the population of lymphocytes related to eradication of the damage. Transcriptome analysis of hepatocytes, and secretome studies of extracellular media from in vitro experiments, allowed us to elucidate the specific molecular mechanisms regulated by EGFR that mediate hepatocyte production of both pro-fibrotic and pro-inflammatory mediators; these have consequences for the deposition of extracellular matrix proteins, as well as for the immune microenvironment. Overall, our study uncovered novel mechanistic insights regarding EGFR kinase-dependent actions in hepatocytes that reveal its key role in chronic liver damage. © 2024 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Imaging of Cardiac Fibrosis: An Update, From the AJR Special Series on Imaging of Fibrosis.

Myocardial fibrosis (MF) is defined as excessive production and deposition of extra-cellular matrix proteins that result in pathologic myocardial remodeling. Three types of MF have been identified: replacement fibrosis from tissue necrosis, reactive fibrosis from myocardial stress, and infiltrative interstitial fibrosis from progressive deposition of nondegradable material such as amyloid. Although echocardiography, nuclear medicine, and CT play important roles in the assessment of MF, MRI is pivotal in the evaluation of MF, with the late gadolinium enhancement (LGE) technique used as a primary end point. The LGE technique focuses on the pattern and distribution of gadolinium accumulation in the myocardium and assists in the diagnosis and establishment of the cause of both ischemic and nonischemic cardiomyopathy. LGE MRI also aids prognostication and risk stratification. In addition, LGE MRI is used to guide the management of patients considered for ablation for arrhythmias. Parametric mapping techniques, including T1 mapping and extracellular volume measurement, allow detection and quantification of diffuse fibrosis, which may not be detected by LGE MRI. These techniques also allow monitoring of disease progression and therapy response. This review provides an update on the imaging of MF, including prognostication and risk stratification tools, electrophysiologic considerations, and disease monitoring.

IR-780 Dye-based Targeting of Cancer-associated Fibroblasts Improves Cancer Immunotherapy by Increasing Intra-tumoral T Lymphocytes Infiltration.

Immune-checkpoint inhibitors (ICIs) against programmed death (PD)-1/PD-L1 pathway immunotherapy have been demonstrated to be effective in only a subset of patients with cancer, while the rest may exhibit low response or may develop drug resistance after initially responding. Previous studies have indicated that extensive collagen-rich stroma secreted by cancer-associated fibroblasts (CAFs) within the tumor microenvironment is one of the key obstructions of the immunotherapy for some tumors by decreasing the infiltrating cytotoxic T cells. However, there is still a lack of effective therapeutic strategies to control the extracellular matrix by targeting CAFs. The enhanced uptake of IR-780 by CAFs was assessed by using in vivo or ex vivo nearinfrared fluorescence imaging, confocal NIR fluorescent imaging, and CAFs isolation testing. The fibrotic phenotype down-regulation effects and in vitro CAFs killing effect of IR-780 were tested by qPCR, western blot, and flow cytometry. The in vivo therapeutic enhancement of anti-PD-L1 by IR-780 was evaluated on EMT6 and MC38 subcutaneous xenograft mice models. IR-780 has been demonstrated to be preferentially taken up by CAFs and accumulate in the mitochondria. Further results identified low-dose IR-780 to downregulate the fibrotic phenotype, while high-dose IR-780 could directly kill both CAFs and EMT6 cells in vitro. Moreover, IR-780 significantly inhibited extracellular matrix (ECM) protein deposition in the peri-tumoral stroma on subcutaneous EMT6 and MC38 xenografts, which increased the proportion of tumor-infiltrating lymphocytes (TILs) in the deep tumor and further promoted anti-PD-L1 therapeutic efficacy. This work provides a unique strategy for the inhibition of ECM protein deposition in the tumor microenvironment by targeted regulating of CAFs, which destroys the T cell barrier and further promotes tumor response to PD-L1 monoclonal antibody. IR-780 has been proposed as a potential therapeutic small-molecule adjuvant to promote the effect of immunotherapy.

Linagliptin Mitigates TGF-β1 Mediated Epithelial-Mesenchymal Transition in Tacrolimus-Induced Renal Interstitial Fibrosis via Smad/ERK/P38 and HIF-1α/LOXL2 Signaling Pathways.

The dipeptidyl peptidase-4 (DPP-4) inhibitors, a novel anti-diabetic medication family, are renoprotective in diabetes, but a comparable benefit in chronic non-diabetic kidney diseases is still under investigation. This study aimed to elucidate the molecular mechanisms of linagliptin's (Lina) protective role in a rat model of chronic kidney injury caused by tacrolimus (TAC) independent of blood glucose levels. Thirty-two adult male Sprague Dawley rats were equally randomized into four groups and treated daily for 28 d as follows: The control group; received olive oil (1 mL/kg/d, subcutaneously), group 2; received Lina (5 mg/kg/d, orally), group 3; received TAC (1.5 mg/kg/d, subcutaneously), group 4; received TAC plus Lina concomitantly in doses as the same previous groups. Blood and urine samples were collected to investigate renal function indices and tubular injury markers. Additionally, signaling molecules, epithelial-mesenchymal transition (EMT), and fibrotic-related proteins in kidney tissue were assessed by enzyme-linked immunosorbent assay (ELISA) and Western blot analysis, immunohistochemical and histological examinations. Tacrolimus markedly induced renal injury and fibrosis as indicated by renal dysfunction, histological damage, and deposition of extracellular matrix (ECM) proteins. It also increased transforming growth factor β1 (TGF-β1), Smad4, p-extracellular signal-regulated kinase (ERK)1/2/ERK1/2, and p-P38/P38 mitogen-activated protein kinase (MAPK) protein levels. These alterations were markedly attenuated by the Lina administration. Moreover, Lina significantly inhibited EMT, evidenced by inhibiting Vimentin and α-smooth muscle actin (α-SMA) and elevating E-cadherin. Furthermore, Lina diminished hypoxia-related protein levels with a subsequent reduction in Snail and Twist expressions. We concluded that Lina may protect against TAC-induced interstitial fibrosis by modulating TGF-β1 mediated EMT via Smad-dependent and independent signaling pathways.

Multi-omics integration highlights the role of ubiquitination in endometriosis fibrosis

BackgroundEndometriosis, characterized by the presence of active endometrial-like tissues outside the uterus, causes symptoms like dysmenorrhea and infertility due to the fibrosis of endometrial cells, which involves excessive deposition of extracellular matrix (ECM) proteins. Ubiquitination, an important post-transcriptional modification, regulates various biological processes in human diseases. However, its role in the fibrosis process in endometriosis remains unclear.MethodsWe employed multi-omics approaches on two cohorts of endometriosis patients with 39 samples. GO terms and KEGG pathways enrichment analyses were used to investigate the functional changes involved in endometriosis. Pearson’s correlation coefficient analysis was conducted to explore the relationship between global proteome and ubiquitylome in endometriosis. The protein expression levels of ubiquitin-, fibrosis-related proteins, and E3 ubiquitin-protein ligase TRIM33 were validated via Western blot. Transfecting human endometrial stroma cells (hESCs) with TRIM33 small interfering RNA (siRNA) in vitro to explore how TRIM33 affects fibrosis-related proteins.ResultsIntegration of proteomics and transcriptomics showed genes with concurrent change of both mRNA and protein level which involved in ECM production in ectopic endometria. Ubiquitylomics distinguished 1647 and 1698 ubiquitinated lysine sites in the ectopic (EC) group compared to the normal (NC) and eutopic (EU) groups, respectively. Further multi-omics integration highlighted the essential role of ubiquitination in key fibrosis regulators in endometriosis. Correlation analysis between proteome and ubiquitylome showed correlation coefficients of 0.32 and 0.36 for ubiquitinated fibrosis proteins in EC/NC and EC/EU groups, respectively, indicating positive regulation of fibrosis-related protein expression by ubiquitination in ectopic lesions. We identified ubiquitination in 41 pivotal proteins within the fibrosis-related pathway of endometriosis. Finally, the elevated expression of TGFBR1/α-SMA/FAP/FN1/Collagen1 proteins in EC tissues were validated across independent samples. More importantly, we demonstrated that both the mRNA and protein levels of TRIM33 were reduced in endometriotic tissues. Knockdown of TRIM33 promoted TGFBR1/p-SMAD2/α-SMA/FN1 protein expressions in hESCs but did not significantly affect Collagen1/FAP levels, suggesting its inhibitory effect on fibrosis in vitro.ConclusionsThis study, employing multi-omics approaches, provides novel insights into endometriosis ubiquitination profiles and reveals aberrant expression of the E3 ubiquitin ligase TRIM33 in endometriotic tissues, emphasizing their critical involvement in fibrosis pathogenesis and potential therapeutic targets.

Oxidative DNA damage promotes vascular aging associated with changes in extracellular matrix-regulating proteins.

Vascular aging is characterized by vessel stiffening, with increased deposition of extracellular matrix (ECM) proteins including collagens. Oxidative DNA damage occurs in vascular aging, but how it regulates ECM proteins and vascular stiffening is unknown. We sought to determine the relationship between oxidative DNA damage and ECM regulatory proteins in vascular aging. We examined oxidative DNA damage, the major base excision repair (BER) enzyme 8-Oxoguanine DNA Glycosylase (Ogg1) and its regulators, multiple physiological markers of aging, and ECM proteomics in mice from 22-72w. Vascular aging was associated with increased oxidative DNA damage, and decreased expression of Ogg1, its active acetylated form, its acetylation regulatory proteins P300 and CBP, and the transcription factor Foxo3a. Vascular stiffness was examined in vivo in control, Ogg1-/-, or mice with vascular smooth muscle cell-specific expression of Ogg1+ (Ogg1) or an inactive mutation (Ogg1KR). Ogg1-/- and Ogg1KR mice showed reduced arterial compliance and distensibility, and increased stiffness and pulse pressure, whereas Ogg1 expression normalised all parameters to 72w. ECM proteomics identified major changes in collagens with aging, and downregulation of the ECM regulatory proteins Protein 6-lysyl oxidase (LOX) and WNT1-inducible-signaling pathway protein 2 (WISP2). Ogg1 overexpression upregulated LOX and WISP2 both in vitro and in vivo, and downregulated Transforming growth factor β1 (TGFb1) and Collagen 4α1 in vivo compared with Ogg1KR. Foxo3a activation induced Lox, while Wnt3 induction of Wisp2 also upregulated LOX and Foxo3a, and downregulated TGFβ1 and fibronectin 1. In humans, 8-oxo-G increased with vascular stiffness, while active OGG1 reduced with both age and stiffness. Vascular aging is associated with oxidative DNA damage, downregulation of major BER proteins, and changes in multiple ECM structural and regulatory proteins. Ogg1 protects against vascular aging, associated with changes in ECM regulatory proteins including LOX and WISP2.